The Caenorhabditis elegans cullin-RING ubiquitin ligase CRL4DCAF-1 is required for proper germline nucleolus morphology and male development.

Cullin-RING ubiquitin ligases (CRLs) are the largest class of ubiquitin ligases with diverse functions encompassing hundreds of cellular processes. Inactivation of core components of the CRL4 ubiquitin ligase produces a germ cell defect in Caenorhabditis elegans that is marked by abnormal globular morphology of the nucleolus and fewer germ cells. We identified DDB1 Cullin4 associated factor (DCAF)-1 as the CRL4 substrate receptor that ensures proper germ cell nucleolus morphology. We demonstrate that the dcaf-1 gene is the ncl-2 (abnormal nucleoli) gene, whose molecular identity was not previously known. We also observed that CRL4DCAF-1 is required for male tail development. Additionally, the inactivation of CRL4DCAF-1 results in a male-specific lethality in which a percentage of male progeny arrest as embryos or larvae. Analysis of the germ cell nucleolus defect using transmission electron microscopy revealed that dcaf-1 mutant germ cells possess significantly fewer ribosomes, suggesting a defect in ribosome biogenesis. We discovered that inactivation of the sperm-fate specification gene fog-1 (feminization of the germ line-1) or its protein-interacting partner, fog-3, rescues the dcaf-1 nucleolus morphology defect. Epitope-tagged versions of both FOG-1 and FOG-3 proteins are aberrantly present in adult dcaf-1(RNAi) animals, suggesting that DCAF-1 negatively regulates FOG-1 and FOG-3 expression. Murine CRL4DCAF-1 targets the degradation of the ribosome assembly factor periodic trptophan protein 1 (PWP1). We observed that the inactivation of Caenorhabditis elegansDCAF-1 increases the nucleolar levels of PWP1 in the germ line, intestine, and hypodermis. Reducing the level of PWP-1 rescues the dcaf-1 mutant defects of fewer germ cell numbers and abnormal nucleolus morphology, suggesting that the increase in PWP-1 levels contributes to the dcaf-1 germline defect. Our results suggest that CRL4DCAF-1 has an evolutionarily ancient role in regulating ribosome biogenesis including a conserved target in PWP1.

Emerging roles for folate receptor FOLR1 in signaling and cancer.

Folates are B vitamins that function in one-carbon metabolism. Folate receptors are one of three major types of folate transporters. The folate receptors FOLR1 and FOLR2 are overexpressed in multiple cancers. The overexpression of FOLR1 is often associated with increased cancer progression and poor patient prognosis. There is emerging evidence that FOLR1 is involved in signaling pathways that are independent of one-carbon metabolism. Recent publications implicate a direct role of FOLR1 in three signaling pathways: JAK-STAT3, ERK1/2, and as a transcription factor. Six other signaling pathways have been proposed to include FOLR1, but these currently lack sufficient data to infer a direct signaling role for FOLR1. We discuss the data that support noncanonical roles for FOLR1, and its limitations.

Subcellular three-dimensional imaging deep through multicellular thick samples by structured illumination microscopy and adaptive optics.

Structured Illumination Microscopy enables live imaging with sub-diffraction resolution. Unfortunately, optical aberrations can lead to loss of resolution and artifacts in Structured Illumination Microscopy rendering the technique unusable in samples thicker than a single cell. Here we report on the combination of Adaptive Optics and Structured Illumination Microscopy enabling imaging with 150 nm lateral and 570 nm axial resolution at a depth of 80 µm through Caenorhabditis elegans. We demonstrate that Adaptive Optics improves the three-dimensional resolution, especially along the axial direction, and reduces artifacts, successfully realizing 3D-Structured Illumination Microscopy in a variety of biological samples.

Developmental Control of the Cell Cycle: Insights from Caenorhabditis elegans.

During animal development, a single fertilized egg forms a complete organism with tens to trillions of cells that encompass a large variety of cell types. Cell cycle regulation is therefore at the center of development and needs to be carried out in close coordination with cell differentiation, migration, and death, as well as tissue formation, morphogenesis, and homeostasis. The timing and frequency of cell divisions are controlled by complex combinations of external and cell-intrinsic signals that vary throughout development. Insight into how such controls determine in vivo cell division patterns has come from studies in various genetic model systems. The nematode Caenorhabditis elegans has only about 1000 somatic cells and approximately twice as many germ cells in the adult hermaphrodite. Despite the relatively small number of cells, C. elegans has diverse tissues, including intestine, nerves, striated and smooth muscle, and skin. C. elegans is unique as a model organism for studies of the cell cycle because the somatic cell lineage is invariant. Somatic cells divide at set times during development to produce daughter cells that adopt reproducible developmental fates. Studies in C. elegans have allowed the identification of conserved cell cycle regulators and provided insights into how cell cycle regulation varies between tissues. In this review, we focus on the regulation of the cell cycle in the context of C. elegans development, with reference to other systems, with the goal of better understanding how cell cycle regulation is linked to animal development in general.

The Energy Maintenance Theory of Aging: Maintaining Energy Metabolism to Allow Longevity.

Fused, elongated mitochondria are more efficient in generating ATP than fragmented mitochondria. In diverse C. elegans longevity pathways, increased levels of fused mitochondria are associated with lifespan extension. Blocking mitochondrial fusion in these animals abolishes their extended longevity. The long-lived C. elegans vhl-1 mutant is an exception that does not have increased fused mitochondria, and is not dependent on fusion for longevity. Loss of mammalian VHL upregulates alternate energy generating pathways. This suggests that mitochondrial fusion facilitates longevity in C. elegans by increasing energy metabolism. In diverse animals, ATP levels broadly decreases with age. Substantial evidence supports the theory that increasing or maintaining energy metabolism promotes the survival of older animals. Increased ATP levels in older animals allow energy-intensive repair and homeostatic mechanisms such as proteostasis that act to prevent cellular aging. These observations support the emerging paradigm that maintaining energy metabolism promotes the survival of older animals.

Dafachronic acid inhibits C. elegans germ cell proliferation in a DAF-12-dependent manner.

Dafachronic acid (DA) is a bile acid-like steroid hormone that regulates dauer formation, heterochrony, and lifespan in C. elegans. Here, we describe that DA is an inhibitor of C. elegans germ stem cell proliferation in adult hermaphrodites. Using a C. elegans germ cell primary culture system, we show that DA inhibits the proliferation of germ cells in vitro. Exogenous DA reduces the frequency of large tumors in adult tumorous germline mutants and decreases the proliferation of wild-type germ stem cells in adult hermaphrodites. In contrast, DA has no appreciable effect on the proliferation of larval-stage germ cells in wild type. The inhibition of adult germ cell proliferation by DA requires its canonical receptor DAF-12. Blocking DA production by inactivating the cytochrome P450 DAF-9 increases germ cell proliferation in wild-type adult hermaphrodites and the frequency of large tumors in germline tumorous mutants, suggesting that DA inhibits the rate of germ cell proliferation under normal growth conditions.

Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants.

The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016.

Increased mitochondrial fusion allows the survival of older animals in diverse C. elegans longevity pathways.

Mitochondria are dynamic organelles that undergo fusion and fission events. Mitochondrial dynamics are required for mitochondrial viability and for responses to changes in bioenergetic status. Here we describe an insulin-signaling and SCFLIN-23-regulated pathway that controls mitochondrial fusion in Caenorhabditis elegans by repressing the expression of the mitochondrial proteases SPG-7 and PPGN-1. This pathway is required for mitochondrial fusion in response to physical exertion, and for the associated extension in lifespan. We show that diverse longevity pathways exhibit increased levels of elongated mitochondria. The increased mitochondrial fusion is essential for longevity in the diverse longevity pathways, as inhibiting mitochondrial fusion reduces their lifespans to wild-type levels. Our results suggest that increased mitochondrial fusion is not a major driver of longevity, but rather is essential to allow the survival of older animals beyond their normal lifespan in diverse longevity pathways.Mitochondria can undergo shape changes as a result of fusion and fission events. Here the authors describe how insulin signalling regulates mitochondrial fusion in C. elegans, and show that mitochondrial fusion is necessary, but not sufficient, for longevity of worms with mutations that increase lifespan.

Addressing a weakness of anticancer therapy with mitosis inhibitors: Mitotic slippage.

Mitosis inhibitors, which include antimicrotubule drugs, are chemotherapy agents that induce the arrest and apoptosis of mitotic cells. Mitotic slippage, in which mitotically arrested cells exit mitosis, limits the effectiveness of mitosis inhibitors. We have discovered that the CRL2ZYG11A/B ubiquitin ligase promotes mitotic slippage. The combination of antimicrotubule drugs and a CRL2ZYG11A/B inhibitor prevents mitotic slippage to increase antimitotic efficacy.

FEM1 proteins are ancient regulators of SLBP degradation.

FEM1A, FEM1B, and FEM1C are evolutionarily-conserved VHL-box proteins, the substrate recognition subunits of CUL2-RING E3 ubiquitin ligase complexes. Here, we report that FEM1 proteins are ancient regulators of Stem-Loop Binding Protein (SLBP), a conserved protein that interacts with the stem loop structure located in the 3' end of canonical histone mRNAs and functions in mRNA cleavage, translation and degradation. SLBP levels are highest during S-phase coinciding with histone synthesis. The ubiquitin ligase complex SCFcyclin F targets SLBP for degradation in G2 phase; however, the regulation of SLBP during other stages of the cell cycle is poorly understood. We provide evidence that FEM1A, FEM1B, and FEM1C interact with and mediate the degradation of SLBP. Cyclin F, FEM1A, FEM1B and FEM1C all interact with a region in SLBP's N-terminus using distinct degrons. An SLBP mutant that is unable to interact with all 4 ligases is expressed at higher levels than wild type SLBP and does not oscillate during the cell cycle. We demonstrate that orthologues of SLBP and FEM1 proteins interact in C. elegans and D. melanogaster, suggesting that the pathway is evolutionarily conserved. Furthermore, we show that FEM1 depletion in C. elegans results in the upregulation of SLBP ortholog CDL-1 in oocytes. Notably, cyclin F is absent in flies and worms, suggesting that FEM1 proteins play an important role in SLBP targeting in lower eukaryotes.

The ubiquitin ligase CRL2ZYG11 targets cyclin B1 for degradation in a conserved pathway that facilitates mitotic slippage.

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is known to target the degradation of cyclin B1, which is crucial for mitotic progression in animal cells. In this study, we show that the ubiquitin ligase CRL2ZYG-11 redundantly targets the degradation of cyclin B1 in Caenorhabditis elegans and human cells. In C. elegans, both CRL2ZYG-11 and APC/C are required for proper progression through meiotic divisions. In human cells, inactivation of CRL2ZYG11A/B has minimal effects on mitotic progression when APC/C is active. However, when APC/C is inactivated or cyclin B1 is overexpressed, CRL2ZYG11A/B-mediated degradation of cyclin B1 is required for normal progression through metaphase. Mitotic cells arrested by the spindle assembly checkpoint, which inactivates APC/C, often exit mitosis in a process termed "mitotic slippage," which generates tetraploid cells and limits the effectiveness of antimitotic chemotherapy drugs. We show that ZYG11A/B subunit knockdown, or broad cullin-RING ubiquitin ligase inactivation with the small molecule MLN4924, inhibits mitotic slippage in human cells, suggesting the potential for antimitotic combination therapy.

Bacterial Folates Provide an Exogenous Signal for C. elegans Germline Stem Cell Proliferation.

Here we describe an in vitro primary culture system for Caenorhabditis elegans germline stem cells. This culture system was used to identify a bacterial folate as a positive regulator of germ cell proliferation. Folates are a family of B-complex vitamins that function in one-carbon metabolism to allow the de novo synthesis of amino acids and nucleosides. We show that germ cell proliferation is stimulated by the folate 10-formyl-tetrahydrofolate-Glun both in vitro and in animals. Other folates that can act as vitamins to rescue folate deficiency lack this germ cell stimulatory activity. The bacterial folate precursor dihydropteroate also promotes germ cell proliferation in vitro and in vivo, despite its inability to promote one-carbon metabolism. The folate receptor homolog FOLR-1 is required for the stimulation of germ cells by 10-formyl-tetrahydrofolate-Glun and dihydropteroate. This work defines a folate and folate-related compound as exogenous signals to modulate germ cell proliferation.

Enhanced resolution through thick tissue with structured illumination and adaptive optics.

Structured illumination microscopy provides twice the linear resolution of conventional fluorescence microscopy, but in thick samples, aberrations degrade the performance and limit the resolution. Here, we demonstrate structured illumination microscopy through 35 µm of tissue using adaptive optics (AO) to correct aberrations resulting in images with a resolution of 140 nm. We report a 60% minimum improvement in the signal-to-noise ratio of the structured illumination reconstruction through thick tissue by correction with AO.

Implications of an absolute simultaneity theory for cosmology and universe acceleration.

An alternate Lorentz transformation, Absolute Lorentz Transformation (ALT), has similar kinematics to special relativity yet maintains absolute simultaneity in the context of a preferred reference frame. In this study, it is shown that ALT is compatible with current experiments to test Lorentz invariance only if the proposed preferred reference frame is locally equivalent to the Earth-centered non-rotating inertial reference frame, with the inference that in an ALT framework, preferred reference frames are associated with centers of gravitational mass. Applying this theoretical framework to cosmological data produces a scenario of universal time contraction in the past. In this scenario, past time contraction would be associated with increased levels of blueshifted light emissions from cosmological objects when viewed from our current perspective. The observation that distant Type Ia supernovae are dimmer than predicted by linear Hubble expansion currently provides the most direct evidence for an accelerating universe. Adjusting for the effects of time contraction on a redshift-distance modulus diagram produces a linear distribution of supernovae over the full redshift spectrum that is consistent with a non-accelerating universe.

Cancer driver candidate genes AVL9, DENND5A and NUPL1 contribute to MDCK cystogenesis.

AVL9, DENND5A and NUPL1 are among the cancer driver candidate genes previously identified via dog-human comparison, and may function in epithelial cell polarity as indicated by bioinformatics analysis. To better understand their cellular functions and roles in cancer, we knocked down each gene in MDCKII cells through shRNA and performed three-dimensional culture. Compared to the control, the knockdown clones developed significantly more abnormal cysts, e.g., cysts with the lumen harboring dead and/or live cells, or cysts having multiple lumens. Further analysis revealed that abnormalities initiated at the first cell division and persisted throughout the entire cystogenesis process. For NUPL1-knockdown cells, abnormal cytogenesis largely arose from faulty cell divisions, notably monopolar spindles or spindles with poorly separated poles. For AVL9- or DENND5A-knockdown cells, abnormalities originated from both aberrant intracellular trafficking and defective mitosis. Moreover, while all knockdown clones displayed an accelerated rate of both cell proliferation and death, only AVL9- and DENND5A-knockdowns, but not NUPL1-knockdown, promoted cell migration. These observations indicate that NUPL1 contributes to bipolar spindle formation, whereas AVL9 and DENND5A participate in both intracellular trafficking and cell cycle progression. Our study shed lights on these genes' normal cellular functions and on how their alteration contributes to carcinogenesis.

C. elegans cell cycle analysis.

Caenorhabditis elegans is an important system for the study of cell cycle regulation in the context of animal development. One of the most powerful features of C. elegans is the invariant cell lineage in which somatic cells initiate cell division at specific times within the developmental program. The cell lineage is fully known and provides the foundation for the analysis of cell cycle progression at single-cell resolution in a multicellular organism. In this chapter, we describe the different types of cell cycles observed in C. elegans, and provide methods and reagents for the analysis of cell cycle progression as well as specific cell cycle phases. We also provide strategies for the analysis and proper interpretation of cell cycle and checkpoint mutants.

Multiple degradation pathways regulate versatile CIP/KIP CDK inhibitors.

The mammalian CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors (CKIs) comprises three proteins--p21(Cip1/WAF1), p27(Kip1), and p57(Kip2)--that bind and inhibit cyclin-CDK complexes, which are key regulators of the cell cycle. CIP/KIP CKIs have additional independent functions in regulating transcription, apoptosis and actin cytoskeletal dynamics. These divergent functions are performed in distinct cellular compartments and contribute to the seemingly contradictory observation that the CKIs can both suppress and promote cancer. Multiple ubiquitin ligases (E3s) direct the proteasome-mediated degradation of p21, p27 and p57. This review analyzes recent data highlighting our current understanding of how distinct E3 pathways regulate subpopulations of the CKIs to control their diverse functions.

Overcoming redundancy: an RNAi enhancer screen for morphogenesis genes in Caenorhabditis elegans.

Morphogenesis is an important component of animal development. Genetic redundancy has been proposed to be common among morphogenesis genes, posing a challenge to the genetic dissection of morphogenesis mechanisms. Genetic redundancy is more generally a challenge in biology, as large proportions of the genes in diverse organisms have no apparent loss of function phenotypes. Here, we present a screen designed to uncover redundant and partially redundant genes that function in an example of morphogenesis, gastrulation in Caenorhabditis elegans. We performed an RNA interference (RNAi) enhancer screen in a gastrulation-sensitized double-mutant background, targeting genes likely to be expressed in gastrulating cells or their neighbors. Secondary screening identified 16 new genes whose functions contribute to normal gastrulation in a nonsensitized background. We observed that for most new genes found, the closest known homologs were multiple other C. elegans genes, suggesting that some may have derived from rounds of recent gene duplication events. We predict that such genes are more likely than single copy genes to comprise redundant or partially redundant gene families. We explored this prediction for one gene that we identified and confirmed that this gene and five close relatives, which encode predicted substrate recognition subunits (SRSs) for a CUL-2 ubiquitin ligase, do indeed function partially redundantly with each other in gastrulation. Our results implicate new genes in C. elegans gastrulation, and they show that an RNAi-based enhancer screen in C. elegans can be used as an efficient means to identify important but redundant or partially redundant developmental genes.

CRL2(LRR-1) targets a CDK inhibitor for cell cycle control in C. elegans and actin-based motility regulation in human cells.

The Cip/Kip CDK inhibitor (CKI) p21(Cip1/WAF1) has a critical role in the nucleus to limit cell proliferation by inhibiting CDK-cyclin complexes. In contrast, cytoplasmic p21 regulates cell survival and the actin cytoskeleton. These divergent functions for p21 in different cellular compartments suggest the necessity for complex regulation. In this study, we identify the CRL2(LRR-1) ubiquitin ligase as a conserved regulator of Cip/Kip CKIs that promotes the degradation of C. elegans CKI-1 and human p21. The nematode CRL2(LRR-1) complex negatively regulates nuclear CKI-1 levels to ensure G1-phase cell cycle progression in germ cells. In contrast, human CRL2(LRR1) targets cytoplasmic p21, acting as a critical regulator of cell motility that promotes a nonmotile stationary cell state by preventing p21 from inhibiting the Rho/ROCK/LIMK pathway. Inactivation of human CRL2(LRR1) leads to the activation of the actin-depolymerizing protein cofilin, dramatic reorganization of the actin cytoskeleton, and increased cell motility.

Intracellular protein glycosylation modulates insulin mediated lifespan in C.elegans.

O-linked-ß-N-acetylglucosamine (O-GlcNAc) modification is a regulatory, nuclear and cytoplasmic post-translational glycosylation of proteins associated with age-related diseases such as Alzheimer's, Parkinson's, and type II diabetes. Global elevation of O-GlcNAc levels on intracellular proteins can induce insulin resistance, the hallmark of type II diabetes, in mammalian systems. InC. elegans, attenuation of the insulin-like signal transduction pathway increases adult lifespan of the nematode. We demonstrate that the O-GlcNAc cycling enzymes OGT and OGA, which add and remove O-GlcNAc respectively, modulate lifespan in C. elegans. Median adult lifespan is increased in an oga-1 deletion strain while median adult life span is decreased upon ogt-1 deletion. The O-GlcNAc-mediated effect on nematode lifespan is dependent on the FoxO transcription factor DAF-16. DAF-16 is a key factor in the insulin-like signal transduction pathway to regulate reproductive development, lifespan, stress tolerance, and dauer formation in C. elegans. Our data indicates that O-GlcNAc cycling selectively influences only a subset of DAF-16 mediated phenotypes, including lifespan and oxidative stress resistance. We performed an affinity purification of O-GlcNAc-modified proteins and observed that a high percentage of these proteins are regulated by insulin signaling and/or impact insulin pathway functional outcomes, suggesting that the O-GlcNAc modification may control downstream effectors to modulate insulin pathway mediated cellular processes.